Epithelial cells have a distinct architecture, including an apico-basally polarized plasma membrane that is critical for their proper function in embryonic development and adult physiology. Defects in epithelial polarity contribute to many human diseases, including congestive heart failure and cystic fibrosis, and have been implicated in the transition between benign and malignant carcinomas. The long-term goal of this project is to understand the cellular and molecular mechanisms that generate epithelial polarity. While studies in cell culture have identified surface cues responsible for initiating epithelial polarization, the identity and mechanism of proteins that regulate the internal cellular machinery, such as the cytoskeleton and protein trafficking pathways, to execute polarity are not known. To identify such regulators we have used genetic screens in Drosophila to search for proteins required for epithelial architecture of the embryonic, larval, or adult stages. Our screens have identified a cell junction-localized protein called Scribble (Scrib) that is essential for the polarity of epithelia at all three stages of development. Scrib also acts as a tumor suppressor, revealing that Scrib links polarity and proliferation control in Drosophila. Scrib encodes a protein with four PDZ protein-protein interaction domains, suggesting that it acts as a scaffolding protein. PDZ domains are found in many proteins implicated in cell polarity, but the mechanism of PDZ-mediated polarization is not known. The experiments in this proposal are designed to uncover the mechanism of Scrib polarizing activity by isolating the proteins with which Scrib interacts to mediate polarity. The aims are: 1) Determine the Scrib interaction domains required for polarity regulation; 2) Biochemically isolate new members of the Scrib complex, and characterize their functions in epithelia; and 3) Genetically identify additional genes in the Scrib pathway as modifiers of Scrib activity. Given the strong conservation of Scrib structure, function, and localization across phylogeny, it is likely that our results will provide new insight into the mechanisms of epithelial polarity in all animals, and inform our understanding of how loss of epithelial cytoarchitecture may contribute to human tumor progression.